Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-29
2003-07-29
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C530S350000, C435S320100, C435S325000, C435S348000, C435S252300, C435S254110
Reexamination Certificate
active
06599717
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to newly identified vascular endothelial growth factor receptor polynucleotides, their encoded polypeptides, and uses and production of such polynucleotides and polypeptides.
Formation of new blood vessels occurs as a result of two processes: vasculogenesis, which is the formation of blood vessels from progenitor cells, and angiogenesis, which is the formation of new blood vessels from preexisting vessels. Vascular endothelial growth factor (VEGF) is a secreted glycoprotein that induces angiogenesis and plays a central role in the regulation of vasculogenesis. It is highly specific for vascular endothelial cells (Dvorak et al., Am. J. Pathol. (1995) 146:1029-1039). VEGF is also known as vascular permeability factor (VPF) because of its permeabilizing effect on blood vessels. In addition to its role in the development of the vascular system, VEGF has been found to be involved in the differentiation of endothelial cells (Carmeliet et al., Nature (1996) 380:435-439; Ferrara et al., Nature, (1996) 380:439-442), cell migration, and apoptosis inhibition.
Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis (Folkman, Nature Med. (1995) 1:27-31) and to the etiology of several additional diseases that are characterized by abnormal angiogenesis, such as metastasis, diabetic retinopathy, rheumatoid arthritis (Ferrera, Breast Cancer Res. Treat. (1995) 127-137), and abnormal wound healing (Brown et al., J. Exp. Med. (1992) 176:1375-1379).
Antibodies against VEGF can suppress tumor growth in vivo (Kim et al., Nature (1993) 362:841-844), indicating that VEGF antagonists could have broad therapeutic applications. VEGF molecules bind tyrosine kinase receptors known as VEGF receptors (VEGFRs). Three high affinity VEGFRs have been characterized in vertebrates, all of which are mainly expressed in vascular endothelial cells: VEGFR-1/FLT-1 (Yoshida et al., Cytogenet. Cell Genet. (1987) 46:724; Fong et al., Nature (1995) 376:65-69), VEGFR-2/KDR/FLK-1 (Terman et al., Oncogene (1991) 6:1677-1683; Matthews etal., Proc. Nat. Acad. Sci., (1991) 88:9026-9030), and VEGFR-3/FLT4 (Alitalo et al., U.S. Pat. No. 5,776,755; Joukov et al., EMBO J., (1996) 15:290-298). VEGFR-1, VEGFR-2, and VEGFR-3 are members of the PDGF (platelet derived growth factor) receptor family (Yarden and Ulirich, Ann. Rev. Biochem. (1988) 57: 443-478). An interesting aspect of current VEGFR biology is the perceived importance of a soluble form of VEGFR (sVEGFR), the extracellular domain without the transmembrane or intracellular domain, as an antagonist of VEGF action.
Members of signaling pathways are used reiteratively throughout evolution. For example, members of the Fibroblast Growth Factor (FGF) pathway are used in the same manner and for the same purpose, namely patterning branching morphogenesis of the respiratory system, by both insects and mammals (Metzger R J, and Krasnow M A, Science (1999) 284:1635-1639). There is a growing body of information regarding the modular subunits and the high-resolution structure of VEGF family members. Several different VEGF genes and their receptors have been identified in vertebrates. Genes from
Caenorhabditis elegans
encoding tyrosine kinase receptors sharing structural features with mammalian VEGFRs have been reported (Popovici et al., 1999 International Worm meeting abstract 680).
There is a clear need for a better understanding of the genetic pathways that VEGF gene family members are involved in. Further knowledge of the genetic pathways that involve or interact with VEGF as well as interacting pathway members and their collective functions and dysfunctions, could be used to develop therapeutics specifically targeted to the disease. The use of invertebrate model organism genetics and related technologies can greatly facilitate the elucidation of biological pathways (Scangos, Nat. Biotechnol. (1997) 15:1220-1221; Margolis and Duyk, Nature Biotech. (1998) 16:311). Invertebrate model organisms can also be used in the screening of putative pharmaceutical agents that are specifically targeted to a gene of interest. The identification of novel VEGF or VEGFR orthologs in model organisms such as
Drosophila melanogaster
would provide tools for genetic and molecular study and validation of these molecules as potential pharmaceutical targets.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide invertebrate homologs of VEGFR that can be used in genetic screening methods to characterize pathways that VEGFR may be involved in as well as other interacting genetic pathways. It is also an object of the invention to provide methods for screening compounds that interact with VEGFR such as those that may have utility as therapeutics.
These and other objects are provided by the present invention which concerns the identification and characterization of a novel VEGFR in
Drosophila melanogaster
, hereinafter referred to as “dmVEGFR”. Isolated nucleic acid molecules are provided that comprise nucleic acid sequences encoding dmVEGFR proteins as well as novel fragments and derivatives thereof. Methods of using the isolated nucleic acid molecules and fragments of the invention are described, such as use of RNA interference methods that block dmVEGFR activity. Vectors and host cells comprising the dmVEGFR nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a dmVEGFR protein.
An important utility of the novel dmVEGFR nucleic acids and proteins is that they can be used in screening assays to identify candidate compounds which are potential therapeutics that interact with dmVEGFR proteins. Further, the extracellular domain of dmVEGFR can be used as a reagent for purification of its ligand, as a biological probe of VEGF function in model organisms, and as an antagonist to VEGFR activity in vitro and in vivo.
Screening assays typically comprise contacting a dmVEGFR protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the dmVEGFR protein. The assays may comprise administering the candidate molecules to cultured host cells that have been genetically engineered to express the dmVEGFR proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism that are genetically engineered to express a dmVEGFR protein.
The genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying dmVEGFR activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the dmVEGFR protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first animal and, additionally has a mutation in a gene of interest. Any difference between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the dmVEGFR protein.
DETAILED DESCRIPTION OF THE INVENTION
The use of invertebrate model organism genetics and related technologies can greatly facilitate the elucidation of biological pathways (Scangos, Nat. Biotechnol. (1997) 15:1220-1221; Margolis and Duyk, supra). Of particular use is the insect model organism,
Drosophila melanogaster
(hereinafter referred to generally as “Drosophila”). An extensive search for Vascular Endothelial Growth Factor Receptor, hereinafter referred to as VEGFR, nucleic acids and their encoded proteins in Drosophila was conducted in an attempt to identify new and useful tools for probing the function and regulation of the VEGFR genes, and for use as targets in drug discovery.
Novel VEGFR nucleic acids and their encoded proteins, hereinafter referred to as dmVEGFR, are identified herein. The newly identified dmVEGFR nucleic acids can be used for the generation of mutant phenotypes in animal models or in living cells that can be used to st
Ebens, Jr. Allen James
Hammonds R. Glenn
Heller Jonathan C.
Weddell Greg
Winslow John W.
Brunelle Jan P.
Exelixis Inc.
Kaufman Claire M.
Shayesteh Laleh
Spector Lorraine
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